Artigo Acesso aberto Revisado por pares

Upstream AUGs in embryonic proinsulin mRNA control its low translation level

2003; Springer Nature; Volume: 22; Issue: 20 Linguagem: Inglês

10.1093/emboj/cdg515

ISSN

1460-2075

Autores

Catalina Hernández‐Sánchez, Alicia Mansilla, Enrique J. de la Rosa, G. Elisabeth Pollerberg, Encarnación Martı́nez-Salas, Flora de Pablo,

Tópico(s)

Pancreatic function and diabetes

Resumo

Article15 October 2003free access Upstream AUGs in embryonic proinsulin mRNA control its low translation level Catalina Hernández-Sánchez Corresponding Author Catalina Hernández-Sánchez Group of Growth Factors in Vertebrate Development, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040 Madrid, Spain Search for more papers by this author Alicia Mansilla Alicia Mansilla Group of Growth Factors in Vertebrate Development, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040 Madrid, Spain Search for more papers by this author Enrique J. de la Rosa Enrique J. de la Rosa Group of Growth Factors in Vertebrate Development, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040 Madrid, Spain Search for more papers by this author G.Elisabeth Pollerberg G.Elisabeth Pollerberg Department of Developmental Neurobiology, Institute of Zoology, University of Heidelberg, Im Neuenheimer Feld 232, D-69120 Heidelberg, Germany Search for more papers by this author Encarna Martínez-Salas Encarna Martínez-Salas Centro de Biología Molecular ‘Severo Ochoa’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author Flora de Pablo Flora de Pablo Group of Growth Factors in Vertebrate Development, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040 Madrid, Spain Search for more papers by this author Catalina Hernández-Sánchez Corresponding Author Catalina Hernández-Sánchez Group of Growth Factors in Vertebrate Development, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040 Madrid, Spain Search for more papers by this author Alicia Mansilla Alicia Mansilla Group of Growth Factors in Vertebrate Development, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040 Madrid, Spain Search for more papers by this author Enrique J. de la Rosa Enrique J. de la Rosa Group of Growth Factors in Vertebrate Development, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040 Madrid, Spain Search for more papers by this author G.Elisabeth Pollerberg G.Elisabeth Pollerberg Department of Developmental Neurobiology, Institute of Zoology, University of Heidelberg, Im Neuenheimer Feld 232, D-69120 Heidelberg, Germany Search for more papers by this author Encarna Martínez-Salas Encarna Martínez-Salas Centro de Biología Molecular ‘Severo Ochoa’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author Flora de Pablo Flora de Pablo Group of Growth Factors in Vertebrate Development, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040 Madrid, Spain Search for more papers by this author Author Information Catalina Hernández-Sánchez 1, Alicia Mansilla1, Enrique J. de la Rosa1, G.Elisabeth Pollerberg2, Encarna Martínez-Salas3 and Flora de Pablo1 1Group of Growth Factors in Vertebrate Development, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040 Madrid, Spain 2Department of Developmental Neurobiology, Institute of Zoology, University of Heidelberg, Im Neuenheimer Feld 232, D-69120 Heidelberg, Germany 3Centro de Biología Molecular ‘Severo Ochoa’, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Cantoblanco, E-28049 Madrid, Spain *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:5582-5592https://doi.org/10.1093/emboj/cdg515 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Proinsulin is expressed prior to development of the pancreas and promotes cell survival. Here we study the mechanism affecting the translation efficiency of a specific embryonic proinsulin mRNA. This transcript shares the coding region with the pancreatic form, but presents a 32 nt extended leader region. Translation of proinsulin is markedly reduced by the presence of two upstream AUGs within the 5′ extension of the embryonic mRNA. This attenuation is lost when the two upstream AUGs are mutated to AAG, leading to translational efficiency similar to that of the pancreatic mRNA. The upstream AUGs are recognized as initiator codons, because expression of upstream ORF is detectable from the embryonic transcript, but not from the mutated or the pancreatic mRNAs. Strict regulation of proinsulin biosynthesis appears to be necessary, since exogenous proinsulin added to embryos in ovo decreased apoptosis and generated abnormal developmental traits. A novel mechanism for low level proinsulin expression thus relies on upstream AUGs within a specific form of embryonic proinsulin mRNA, emphasizing its importance as a tightly regulated developmental signal. Introduction Insulin is considered an essential anabolic hormone, produced and secreted into the blood by the adult pancreas. In chickens and in humans, proinsulin is encoded by a single gene (Perler et al., 1980). The presence of proinsulin mRNA variants differing in their 5′ untranslated regions (5′ UTRs) was recently demonstrated. In chick embryos, we found a novel proinsulin transcript generated by alternative transcription start sites whose synthesis is developmentally regulated (Hernández-Sánchez et al., 2002). In humans, an intron 1 splice variant is metabolically regulated and increases translation efficiency (Shalev et al., 2002). In adult organisms, insulin biosynthesis by pancreatic β-cells is a paradigm of tissue-specific expression. The mature β-cells synthesize the ∼9 kDa proinsulin precursor, which is processed to insulin (∼6 kDa) by the endopeptidases PC2 and PC3 in the secretory granules. Circulating insulin is responsible for maintaining glucose homeostasis, and plasma hormone levels are largely dependent on food intake. Glucose is, in turn, the central regulator of pancreatic insulin biosynthesis at multiple steps (Robbins et al., 1984; Rhodes, 2000). Plasma glucose concentration controls insulin mRNA levels by stimulating insulin gene transcription, as well as insulin mRNA processing and stability (Brunstedt and Chan, 1982; Nielsen et al., 1985; Welsh et al., 1985; Leibiger et al., 1998); in addition, glucose modulates insulin protein synthesis initiation and elongation rates (Permutt, 1974; Itoh and Okamoto, 1980; Welsh et al., 1986). In the past decade, several observations have suggested that low levels of prepancreatic and extrapancreatic proinsulin expression displayed physiological functions (reviewed in De Pablo and Roth, 1990; De Pablo and de la Rosa, 1995; Varela-Nieto et al., 2003). Proinsulin expression during early development occurs prior to formation of the pancreatic primordium in several vertebrates (Serrano et al., 1989; Shuldiner et al., 1991; Deltour et al., 1993). Indeed, insulin-related molecules are expressed and control vital functions in organisms without a pancreas, such as nematodes (Pierce et al., 2001) and flies (Brogiolo et al., 2001). In the neurulating chick embryo and retina (a part of the central nervous system), proinsulin mRNA is expressed and translated to proinsulin. In contrast to the pancreatic protein, this proinsulin remains unprocessed, due to the absence PC2 convertase, and is rapidly secreted (Hernández-Sánchez et al., 1995, 2002; Morales et al., 1997; Alarcón et al., 1998; Díaz et al., 1999). In the chick, embryonic proinsulin mRNA levels are ∼1000-fold lower than those of the postnatal pancreas (Serrano et al., 1989), but are nevertheless essential for correct development. Proinsulin, present at embryonic stages when insulin-like growth factor I (IGF-I) is very low, promotes cell survival during neurulation and retinal neurogenesis. Interference with proinsulin and insulin-receptor biosynthesis by antisense oligonucleotides, as well as blockade of proinsulin signalling by anti-insulin and anti-insulin receptor antibodies, induces apoptosis in early chick embryos (De Pablo et al., 1985a; Morales et al., 1997; Díaz et al., 2000; Hernández-Sánchez et al., 2002). Proinsulin gene products appear to have a dual biological function. First, as a tissue factor in early development, low local proinsulin levels are essential to regulate programmed cell death, a highly-regulated physiological process required for correct body pattern formation and development (reviewed by Jacobson et al., 1997; Vaux and Korsmeyer, 1999; de la Rosa and De Pablo, 2000). In postnatal organisms, mature insulin assumes its much better-characterized hormonal role, acting on newly acquired target tissues such as adipose tissue. At that stage, the pancreas functions as an insulin ‘factory’ (Orci et al., 1988), able to respond to rapidly changing metabolic requirements. In parallel with these very distinct functions, we found different embryonic and pancreatic proinsulin transcripts (Hernández-Sánchez et al., 2002). Embryonic proinsulin mRNA shares its coding region with the pancreatic form, but has an extended 5′ UTR. In addition, embryonic proinsulin mRNA levels are developmentally regulated, but are not glucose dependent, a difference from pancreatic mRNA (Pérez-Villamil et al., 1994), suggesting distinct regulation of expression. Pancreatic proinsulin mRNA shows the typical 5′ UTR of higher eukaryotic genes, as it is short, unstructured and without additional AUGs. In contrast, genes that display critical proliferation or survival functions, such as oncogenes, cytokines, signal transduction proteins and transcription factors, harbour upstream AUGs (upAUGs) and associated open reading frames (upORF) (Sachs and Geballe, 2000; Morris and Geballe, 2000; Kozak 2002a). Here we characterize a novel form of control of the proinsulin gene expression by generating alternative transcript structure. In the specific embryonic form, proinsulin translation initiation is stringently repressed by the presence of two upAUGs within the extended 5′ UTR. In ovo, proinsulin addition to chick embryos modulates apoptosis and morphogenesis, suggesting that fine control of the specific embryonic proinsulin transcript structure is part of the regulated signalling required for normal embryo development in vertebrates. Results Embryonic and pancreatic proinsulin transcription initiation sites are adjacent in the genome We recently reported that embryonic proinsulin mRNA shows a 32-nt extension in its 5′ UTR, whereas the coding region is shared with the pancreatic mRNA (Hernández-Sánchez et al., 2002). These alternative transcription start sites mapped in adjacent positions in the genome, as demonstrated by sequencing of genomic PCR products (Figure 1A and B). Sequence analysis of the embryonic proinsulin 5′ UTR (Figure 1C) revealed an upORF with two potential start codons (upAUG1/2) within the 32-nt extension of this mRNA. The first upAUG is located 12 nt upstream of the second, and the upORF overlaps 183 nt with the proinsulin ORF, which is shifted by −1 nt in its phase. Notably, upATG1/2 as well as the proinsulin initiator codon are located in good Kozak consensus sequence (Kozak, 1986). Figure 1.Characterization of pancreatic and embryonic proinsulin leader regions. PCR of the 5′ region of the chicken proinsulin genomic locus (A). Genomic organization of the amplified products (B) according to sequence analysis of the chicken proinsulin gene 5′ region (C). Arrows indicate embryonic and pancreatic transcription start sites; exon (E), intron (I) and primer (P) locations are shown. The proinsulin cDNA and predicted amino acid sequence (C) contain the proinsulin ATG (bold capitals), as well as the two upATGs (bold lowercase) and predicted upORF. Note that upORF is out of the proinsulin reading frame. Download figure Download PowerPoint Embryonic and pancreatic proinsulin mRNAs show distinct translational activity The presence of upAUGs and upORF in embryonic proinsulin mRNA suggests possible differential translational efficiency of this transcript compared with the pancreatic mRNA. We therefore studied the translational activity of embryonic and pancreatic proinsulin mRNAs in vitro and in vivo. To facilitate detection of proinsulin, the embryonic or the pancreatic proinsulin cDNAs were fused to the V5 epitope tag in constructs termed pCPro1B (embryonic) and pCPro1A (pancreatic). Transcription–translation assay of these constructs in a reticulocyte lysate system rendered a 13 kDa protein common to the embryonic and pancreatic constructs that corresponded to the proinsulin V5-tagged protein, as verified by anti-V5 immunoprecipitation (Figure 2A). The amount of proinsulin generated by the embryonic construct (pCPro1B) was nonetheless lower than that generated by the pancreatic construct (pCPro1A). Once it was confirmed that both transcripts were able to generate proinsulin in vitro (also in an un-coupled transcription and translation system, not shown), the differences observed in translation levels were analysed in vivo. NIH3T3 cells were transfected with each of the constructs and proinsulin-V5 was detected by western blot. Untransfected cells or cells transfected with an unrelated construct showed no V5 immunoreactivity (not shown). The amount of proinsulin generated with the embryonic construct was much lower than that in cells transfected with the pancreatic construct (Figure 2B). To assess whether the dramatic difference in proinsulin expression was mediated by translational efficiency or by differences in mRNA levels, the integrity and abundance of the transcripts were analysed by northern blot. Both embryonic and pancreatic proinsulin constructs were expressed at comparable levels relative to endogenous GAPDH mRNA (Figure 2C), suggesting that the differential embryonic and pancreatic proinsulin expression is regulated at a postrancriptional level. Figure 2.Translation of embryonic and pancreatic proinsulin. In vitro-coupled transcription–translation and immunoprecipitation with anti-V5 epitope antibody of embryonic (pCPro1B) and pancreatic (pCPro1A) proinsulin constructs (A). The same constructs were transfected into NIH3T3 cells. Transfected cells were analysed for proinsulin expression by western blotting using anti-V5 epitope antibody (B). β-tubulin was used as a loading control (bottom panel). Northern blot of NIH3T3 transfected cells (C). Membranes were probed for proinsulin (top panel) and GAPDH (bottom panel) mRNA. Proinsulin and GAPDH mRNA levels were quantified by a Fuji image analyzer and proinsulin mRNA levels corrected by that of endogenous GAPDH mRNA levels (proinsulin mRNA/GAPDH mRNA, 1.01 for embryonic and 1 for pancreatic constructs). Download figure Download PowerPoint The good Kozak consensus of the upAUGs suggests that they mediate translation initiation. To examine upORF expression, we generated an antibody against a C-terminal peptide of the upORF. An ∼10 kDa protein was detected by western blot of NIH3T3 cells transfected with the embryonic, but not with the pancreatic, construct (Figure 3A). A single band was observed in standard gels whereas in long gels from experiments in vitro a doublet could be resolved (not shown). Immunostaining with anti-V5 and anti-upORF demostrated cytoplasmic expression of both proinsulin and upORF in the same cells transfected with the embryonic construct whereas only proinsulin was detected in cells transfected with the pancreatic construct (Figure 3B). Figure 3.Expression of upORF in transfected cells. Western blot of NIH3T3 transfected cells with the embryonic (pCPro1B) and pancreatic (pCPro1A) proinsulin constructs using anti-V5 epitope or anti-upORF antibodies for proinsulin and upORF expression, respectively (upper and middle panels) (A). β-tubulin was used as a loading control (bottom panel). Immunostaining of NIH3T3 transfected cells with pCPro1B and pCPro1A constructs using anti-V5 epitope antibody in red and anti-upORF antibody in green (B). Images were captured by a confocal microscope with a 40× objective. Download figure Download PowerPoint Proinsulin is expressed in immortalized embryonic neuroepithelial cells The embryonic proinsulin transcript is translated to a lesser extent than the pancreatic form in the reticulocyte lysate system and in the NIH3T3 cell line. To confirm these observations and relate them to physiological function, we used a cellular system that reflects embryonic proinsulin expression and cell response. Quail RTC5 is a retinal cell line originated from quail embryonic retina undergoing neurogenesis, immortalized by retroviral transduction with the myc oncogene. Under controlled conditions, proliferating cells can be induced to differentiate into cells with mature neuronal characteristics (Pollerberg and Eickholt, 1995; Pollerberg et al., 1995). Embryonic proinsulin mRNA expression was compared in RTC5 cells with incubation day 1.5 (E1.5) whole chick embryos undergoing neurulation. Proinsulin mRNA was characterized by RT–PCR of total RNA using primers directed to the proinsulin coding region (P4, P5) or primers that specifically amplified the embryonic proinsulin mRNA variant (P8, P5). Gel analysis and sequencing (not shown) of the PCR products demonstrated the presence of the embryonic proinsulin transcript in RTC5 cells (Figure 4A). Figure 4.Expression and effect of proinsulin in RTC5 cells. Proinsulin amplification products obtained by RT–PCR of total RNA from E1.5 embryos and RTC5 cells (A). −RT, control without reverse transcriptase. Location of primers (P) used is shown in the upper panel. Apoptosis of RTC5 cells (B). Apoptotic cells were scored by DAPI staining of pyknotic nuclei after 20 h growing in complete medium (serum) or serum-free medium supplemented with the indicated proinsulin concentrations. A minimum of 1000 cells was counted per experimental point. Results represent the mean ± SEM of three replicates. *P ⩽ 0.001 with respect to non-proinsulin treatment. Download figure Download PowerPoint Proinsulin attenuates programmed cell death in the neurulating embryo and developing retina (Díaz et al., 2000; Hernández-Sánchez et al., 2002); we therefore analysed the anti-apoptotic effect of proinsulin on RTC5 cells cultured in serum-free medium. As predicted, proinsulin rescued RTC5 cells from growth factor deprivation-induced apoptotic cell death in a dose-dependent manner. In the presence of 10−7 M proinsulin, the percentage of apoptotic cells decreased from 3.6 to 1.8%, close to the level observed in the presence of serum (1.1%; Figure 4B). In contrast to the antiapoptotic effect, proinsulin addition produced only a slight, non-significant increase in total cell number, even at the highest concentration. Serum starvation for long periods (up to 32 h) did not induce apoptosis in NIH3T3 cells (not shown). All together these data validate the use of RTC5 cells as a model to study regulation of embryonic proinsulin. The embryonic proinsulin mRNA leader region determines translation efficiency Translation of the embryonic proinsulin mRNA yielded a reduced amount of proinsulin concomitant with the synthesis of a polypeptide generated from the upORF. It is therefore possible that translation attenuation was the consequence of the primary 5′ UTR sequence or the presence of the polypeptides originated at upAUG1/2. To determine whether the 5′ leader region was responsible for this effect, we analysed chimeric constructs containing either the embryonic (pGLuc1B) or pancreatic (pGLuc1A) proinsulin 5′ UTRs fused to the luciferase ORF. In this scheme, the upORF encoded in the embryonic chimeric construct differs at the C-terminal end in sequence and length from the upORF encoded in the proinsulin cDNA (compare Figures 5A and 1C). A strong reduction in luciferase activity was observed in both NIH3T3 and RTC5 cells transfected with the embryonic chimeric construct, but not with the pancreatic chimera (Figure 5B). The pancreatic chimera construct was translated efficiently, showing even higher luciferase activity than the luciferase control vector lacking proinsulin 5′ UTR sequences in the NIH3T3 cells, an effect that was not seen in RTC5 cells (Figure 5B). Similar results were obtained when luciferase protein was detected by western blot (Figure 5C). Additionally, pulse labelling of pGLuc1B-transfected NIH3T3 cells consistently showed a lower luciferase translation rate than the pancreatic or control constructs (Figure 5D). Figure 5.Influence of embryonic and pancreatic proinsulin leader region on luciferase chimeric construct translation. Nucleotide and amino acid sequences of the luciferase chimeric constructs (A). The luciferase ATG (bold uppercase), as well as the two upATG (bold lowercase) are labelled. Note that upORF is out of the luciferase reading frame. NIH3T3 and RTC5 cells were transfected with control (pGL3C), embryonic (pGLuc1B) or pancreatic (pGLuc1A) firefly luciferase expression constructs (B). Firefly luciferase (Fluciferase) activity was corrected by Renilla luciferase activity, cotransfected to normalize transfection efficiency. Results represent the mean ± SEM of six samples. Luciferase western blot of NIH3T3 cells transfected with pGL3C, pGLuc1B and pGLuc1A constructs (C). β-tubulin was used as a loading control (bottom panel). [35S]Met/Cys pulse labelling of NIH3T3 cells transfected with the indicated constructs. Cell lysates were immunoprecipitated with anti-luciferase antibody, separated in a polyacrylamide gel and visualized using a Fuji image analyser (D). Download figure Download PowerPoint To study whether the dramatic variance in luciferase activity was mediated by differences in transcription levels and/or mRNA stability, the abundance of the transcripts were analysed by northern blot in transfected NIH3T3 cells. Both embryonic and pancreatic luciferase chimeric constructs were expressed as a single band of about 2 kb, at comparable levels relative to endogenous GAPDH mRNA (Figure 6A and B). To examine the mRNA degradation rate, we treated transfected NIH3T3 cells with the RNA polymerase II inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) for different periods of time. The decay curves for both constructs were identical (Figure 6C), indicating that the embryonic leader region does not influence mRNA degradation levels. Similar results were obtained by inhibiting transcription with actinomycin D (not shown). The GAPDH mRNA degradation rate showed no general transcript degradation in the presence of DRB, albeit the luciferase mRNA was less stable than the GAPDH mRNA. All together, these results point to translational inhibition mediated by the embryonic proinsulin mRNA leader region. Figure 6.mRNA levels and stability of luciferase chimeric constructs with embryonic or pancreatic proinsulin leader regions. Northern blot of NIH3T3 cells transfected with the embryonic (pGLuc1B) or pancreatic (pGluc1A) firefly luciferase expression constructs (A). Membranes were probed for luciferase (top panel) and GAPDH (bottom panel) mRNA. Luciferase and GAPDH mRNA levels were quantified by a Fuji image analyser and luciferase mRNA levels corrected by that of endogenous GAPDH mRNA levels (B). Results represent the mean ± range of two experiments. The RNA polymerase II inhibitor DRB (100 μM) was added 48 h after transfection. RNA was extracted at the times indicated and hybridized in a northern blot with a luciferase probe (C). The luciferase mRNA decay curves are shown after Fuji image analyser quantitation (top panel). The filter was rehybridized with a GAPDH probe (bottom panel). An identical GAPDH mRNA decay curve was obtained for cells transfected with the pGluc1A construct. Download figure Download PowerPoint A low level of embryonic proinsulin mRNA translation depends on the presence of upAUGs Increasing evidence suggests that upAUGs and upORFs significantly influence mRNA translational efficiency (for review see Geballe and Sachs, 2000; Morris and Geballe, 2000; Dever, 2002). The two upAUGs in the 32-nt extension of the embryonic proinsulin mRNA were thus candidates to mediate the low level of translation characterized for the embryonic proinsulin transcript. As hypothesized above, mutation of the two upAUG initiation codons to AAG (Figure 7A) abolished the inhibitory effect caused by the embryonic proinsulin mRNA leader region. The mutated embryonic construct (pCPro1Bmt) produced proinsulin levels comparable to those yielded by the pancreatic construct in the three experimental assays, the reticulocyte lysate system, as well as the transfected NIH3T3 and RTC5 cells (Figure 7B and C). Identical results were obtained with the luciferase constructs in transfected cells. The mutated embryonic chimeric construct (pGLuc1Bmt) rendered luciferase activity comparable to that of the control vector and much higher than the embryonic chimeric construct (Figure 7D). Moreover, expression of the upORF in the embryonic construct was abolished when the two upAUG were mutated to AAG as observed by western blotting (Figure 8) and immuno cytochemistry staining of transfected cells (not shown). Figure 7.Translational effect of upAUG mutation in the embryonic proinsulin leader sequence. Nucleotide sequence of the mutated embryonic proinsulin leader region (A). The proinsulin ATG (bold uppercase) and the two mutated AAG (bold lowercase) are labelled. The mutated nucleotides are underlined. In vitro translation and immunoprecipitation with anti-V5 epitope antibody of pancreatic (pCPro1A), embryonic (pCPro1B) and mutated embryonic (pCPro1Bmt) proinsulin expression constructs (B). The same constructs were transfected into NIH3T3 and RTC5 cells, and proinsulin expression analysed by western blotting. β-tubulin was used as a loading control (bottom panel) (C). NIH3T3 and RTC5 cells were transfected with control (pGL3C), pancreatic (pGLuc1A), embryonic (pGLuc1B) or mutated embryonic (pGLuc1Bmt) firefly luciferase expression constructs (D). Firefly luciferase (Fluciferase) activity was corrected by that of Renilla, cotransfected to normalize transfection efficiency. Results represent the mean ± SEM of four replicates. Download figure Download PowerPoint Figure 8.Effect of upAUG mutation in upORF expression. NIH3T3 cells were transfected with pancreatic (pCPro1A), embryonic (pCPro1B) and mutated embryonic (pCPro1Bmt) proinsulin expression constructs. Cell extracts were analysed by western blotting using anti-V5 epitope or anti-upORF antibodies for proinsulin and upORF expression, respectively (upper and middle panels). β-tubulin was used as a loading control (bottom panel). Download figure Download PowerPoint Proinsulin excess decreases naturally occurring apoptosis and causes embryonic malformations Thus far, the results demonstrate that the embryonic proinsulin transcript presents low translational activity due to two upAUGs not found in pancreatic proinsulin mRNA. To support the physiological relevance of these observations, we tried to confirm in vivo that an excess of proinsulin is as deleterious as its deficit, shown in the past in neurulating embryos (Morales et al., 1997). The effect of moderate addition of exogenous proinsulin was evaluated in neurulating chick embryos. Proinsulin at 10−7 M signals through hybrid insulin/IGF-I receptors and homodimeric insulin receptors, but not through IGF-I receptors (García-de Lacoba et al., 1999; see Discussion for details). Strikingly, gross developmental abnormalities were observed after 8 h treatment in most (12 of 15) proinsulin-treated embryos, but none of the control embryos (Table I). Developmental progression, as measured by number of new somites formed during treatment, was nonetheless identical in control and proinsulin-treated embryos (six to seven somites). Frequent abnormalities included neural defects such as asymmetric anterior neuropore closure, collapsed optic vesicles and flexures in the neural tube (Figure 9B and F). Proinsulin-treated embryos showed a strong reduction in TdT-mediated dUTP nick end labelling (TUNEL) staining in the regions in which malformations had been observed, compared with control embryos (Figure 9C, D, G and H). This concurs with the effect described for proinsulin in attenuation of cell death at this stage (Morales et al., 1997; Hernández-Sánchez et al., 2002). Figure 9.In ovo treatment with exogenous proinsulin. Embryos were treated in ovo with 50 μl of vehicle (A, C, E and G) or with 50 μl 10−7 M proinsulin (B, D, F and H). After 8 h incubation, the morphological aspect of the embryos was evaluated by counterstaining with neutral red (A, B, E and F). Apoptotic cells were visualized by TUNEL staining of whole embryos (C, D, G and H). Serial images were captured in a confocal microscope every 10 μm with a 10× objective and compiled. The bright dots represent TUNEL-stained pyknotic bodies. Different regions shown are boxed in the whole embryo picture (I); the upper box corresponds to the prosencephalon region (A–D) and the lower box to neural tube and rostral somites (E–H). A few dots around the prosencephalon (C) and the somites (G) indicated by asterisks in the boxes (I) are apoptotic cells in the extraembryonic membrane

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